The use of renewable biological material for the production of biofuels is generally motivated by decreasing climate change impacts, by securing the supply of fuels and by economic factors. Lipids are used as a raw material for the production of transportation biofuels, such as biodiesel or renewable diesel, or as their components, for their high energy content and availability. Current raw materials for biodiesel and renewable diesel include vegetable oils, animal fats or recycled greases. Since algae and some other microorganisms are known to naturally produce a wide range of lipids, their use as the source of lipids for biodiesel has been suggested. These microorganism based oils are often called as single cell oils. Lipids are essential for the cell as membrane components, enzyme co-factors, and carbon and energy storages. Some of these lipids, especially triacylglycerols (TAGs), are suitable for biodiesel and/or renewable diesel production.
Many challenges faced with agriculture based raw material, such as vegetable oils. for biodiesel and/or renewable diesel can be reduced with microbial biofuel production. Autotrophic algae are suggested to have significantly higher annual lipid yields per hectare than best oil crops. Alternative option to photosynthetic (autotrophic) production of lipids by algae or cyanobacteria is to utilize heterotrophic microorganisms which produce lipids from organic molecules (such as sugars) without need for light. Importantly, heterotrophic microorganisms can utilize various organic wastes and residues as raw materials for lipid production. Lipid production process using heterotrophic microorganisms comprises cultivating microorganisms in aerated bioreactors, allowing cells to accumulate lipids, harvesting lipid-rich cells and recovering oil from cells.
Single-cell oils have traditionally been used as special products e.g. in health foods, not as commodity chemicals. In these kinds of single cell oil production processes product volumes are relatively small and the product is expensive. Therefore, the cost structure of these processes allows the utilization of expensive feed raw materials and unit operations. Similar kind of production process has also been described for the production of lipids for biodiesel production (Ratledge and Cohen 2008). However, as the product is an inexpensive commodity chemical, the process costs should not be on the level of the process costs of special products. When lipids are used for example as starting material for biodiesel or renewable diesel, it is important that the lipid production process is efficient in terms of lipid yield from substrate and lipid production rate. Since the typical lipid yield by heterotrophic microorganisms is less than 20% weight percent of the fed sugar, the price of raw material has an essential role in cost structure. Since the economy of the production of single cell oils for biofuels is of key importance, new cost-effective processes for lipid production for biofuel production are still of growing interest. Further, the development of more efficient lipid producing organisms is of high interest.
Some bacteria can produce storage lipids, which can be utilized for example as starting material for biodiesel or renewable diesel production. Storage lipids are free fatty acids, acylglycerols, and wax esters and intermediate products thereof. The synthesis of storage lipids is a regulated process in bacteria. The production of storage lipids can be made more efficient by making some genes in the genome of the bacteria inactive and/or by overexpressing some genes in the genome of the bacteria. Such genes have been described for example in the patent publications WO2009/009391 and WO2008/119082. WO2009/009391 discloses a method for making a fatty esters using impure or unpurified alcohol in the production. The recombinant cell used in the method lacks a nucleic acid sequence encoding acyl-CoA dehydrogenase enzyme or the expression of said enzyme is attenuated. The host may comprise also exogenous genes encoding thioesterase, wax synthase, alcohol acetyltransferase, fatty alcohol forming acyl-CoA-reductase, an ester synthase enzyme, or acyl-CoA synthase enzyme. WO2008/119082 discloses recombinant cells from various microorganism hosts expressing or over-expressing gene or genes encoding fatty acid derivative enzymes and a gene encoding an acyl-CoA dehydrogenase enzyme, which gene is modified such that expression of the gene is attenuated.
Various patent publications describe the expression of some genes of the lipid synthesis pathway. WO2008/113041 discloses cracking methods for producing low molecular weight hydrocarbons from biocrude or hydrocarbon feedstock, which may be produced from a recombinant microorganism. The recombinant microorganism may be engineered to express or overexpress peptides, for example acyl-CoA synthetase, thioesterase, acetyl-CoA carboxylase or acyl-carrier protein. WO2007/136762 discloses genetically engineered microorganisms that are capable of synthesizing products derived from the fatty acid biosynthetic pathway (fatty acid derivatives).
Furthermore, DE102004052115 discloses a microorganism comprising a nucleic acid molecule encoding procaryotic acyltransferase, a nucleic acid molecule encoding pyruvatdecarboxylase and a nucleic acid molecule encoding alcoholdehydrogenase.
US 2003145350 discloses a method for increasing the content of short or middle chain length fatty acids in microorganisms and for production of fatty acids and oils having an increased content of short or middle chain length fatty acids. The method comprises the expression of the acyltransferase KAS III in a microorganism.
Although some publications disclose improved microorganism strains for lipid production there is still a need for more efficient processes for lipid production in order to obtain affordable raw material for biofuel and other applications.